Mononuclear non-heme iron active sites are present in a wide range of enzymes involved in a variety of important biological functions requiring dioxygen. These include the lipoxygenases (fatty acid hydroperoxidation), bleomycin (anticancer drug involved in DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), tetrahydropterin dependent hydroxylases (phenylalanine metabolism), and the alpha-ketoglutarate-dependent enzymes (substrate hydroxylation and ring closure). Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class, and substrate and oxygen bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. The general goals of this research program have been to develop new spectroscopic methods for the investigation of non-heme iron active sites and to apply these methods to the study of the above enzymes to obtain molecular level insight into the catalytic mechanisms and to understand the differences in the active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the similarities and differences between non-heme and heme iron sites. Studies thus far have emphasized variable-temperature variable-field magnetic circular dichroism (VTVH MCD) combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme ferrous and ferric sites and to define the unusual electronic structure of the NO complex of non-heme ferrous sites and its relation to possible oxygen intermediates. The specific aims of this proposal are to: l) Complete the development of VTVH MCD as a powerful probe of non-heme ferrous active sites; 2) Develop Fe L- edge X-ray absorption spectroscopy as a new probe of non-heme iron active sites, particularly for ferric centers and oxygen intermediates; 3) Extend studies on lipoxygenases to correlate to the two conflicting crystal structures and to the mammalian enzymes with systematic mutations which influence reactivity, define active site intermediates, and probe the interaction of the active site with inhibitors: 4) Continue studies on bleomycin to understand each of the steps of the catalytic mechanism, determine the effect on the iron site due to the interaction with DNA, and define the relation of bleomycin to heme and other non-heme iron systems; 5) Correlate results on the extradiol dioxygenases with parallel data on the intradiol dioxygenases to determine differences in substrate-iron active site interactions which relate to differences in activation and could influence the position of ring cleavage; 6) Define the interaction of the ferrous site of phenylalanine hydroxylase with the pterin cofactor, probe the key steps of the catalytic mechanism, and determine how mutations which affect the enzyme's reactivity change the active site and its interactions with cofactor and substrate; 7) Understand the interaction of the ferrous site of clavaminate synthase with the alpha-ketoglutarate cofactor, define the interaction of this binary complex with O2 and analogs, and determine the interactions of this site with different substrates which lead to hydroxylation versus ring closure chemistry.